Acc gene

ABSTRACT

The present invention relates to a gene useful in a process to increase the microbial production of carotenoids. The carotenoids astaxanthin is distributed in a wide variety of organisms such as animals, algae and microorganisms. It has a strong antioxidation property against reactive oxygen species. Astaxanthin is used as a coloring reagent, especially in the industry of farmed fish, such as salmon, because astaxanthin imparts distinctive orange-red coloration to the animals and contributes to consumer appeal in the marketplace.

The present invention relates to a gene useful in a process to increasethe microbial production of carotenoids.

The carotenoid astaxanthin is distributed in a wide variety of organismssuch as animals, algae and microorganisms. It has a strong antioxidationproperty against reactive oxygen species. Astaxanthin is used as acoloring reagent, especially in the industry of farmed fish, such assalmon, because astaxanthin imparts distinctive orange-red coloration tothe animals and contributes to consumer appeal in the marketplace.

One of the first steps in the carotenogenic pathway of, e.g. Phaffiarhodozyma, is the condensation of two molecules of acetyl-CoA.Acetyl-CoA is also the substrate for acetyl-CoA carboxylase, one of theenzymes involved in fatty acid biosynthesis.

In one aspect, the present invention provides a novel DNA fragmentcomprising a gene encoding the enzyme acetyl-CoA carboxylase.

More particularly, the present invention provides a DNA containingregulatory regions, such as promoter and terminator, as well as the openreading frame of acetyl-CoA carboxylase gene.

The present invention provides a DNA fragment encoding acetyl-CoAcarboxylase in P. rhodozyma. The said DNA means a cDNA which containsonly open reading frame flanked between the short fragments in its 5′-and 3′-untranslated region, and a genomic DNA which also contains itsregulatory sequences such as its promoter and terminator which arenecessary for the expression of the acetyl-CoA carboxylase gene in P.rhodozyma.

Accordingly, the present invention relates to a polynucleotidecomprising a nucleic acid molecule selected from the group consistingof:

(a) nucleic acid molecules encoding at least the mature form of thepolypeptide depicted in SEQ ID NO:3;

(b) nucleic acid molecules comprising the coding sequence as depicted inSEQ ID NO:2;

(c) nucleic acid molecules whose nucleotide sequence is degenerate as aresult of the genetic code to a nucleotide sequence of (a) or (b);

(d) nucleic acid molecules encoding a polypeptide derived from thepolypeptide encoded by a polynucleotide of (a) to (c) by way ofsubstitution, deletion and/or addition of one or several amino acids ofthe amino acid sequence of the polypeptide encoded by a polynucleotideof (a) to (c);

(e) nucleic acid molecules encoding a polypeptide derived from thepolypeptide whose sequence has an identity of 56.3 % or more to theamino acid sequence of the polypeptide encoded by a nucleic acidmolecule of (a) or (b);

(f) nucleic acid molecules comprising a fragment or a epitope-bearingportion of a polypeptide encoded by a nucleic acid molecule of any oneof (a) to (e) and having acetyl-CoA carboxylase activity;

(g) nucleic acid molecules comprising a polynucleotide having a sequenceof a nucleic acid molecule amplified from Phaffia or Xanthophylomycesnucleic acid library using the primers depicted in SEQ ID NO:4, 5, and6;

(h) nucleic acid molecules encoding a polypeptide having acetyl-CoAcarboxylase activity, wherein said polypeptide is a fragment of apolypeptide encoded by any one of (a) to (g);

(i) nucleic acid molecules comprising at least 15 nucleotides of apolynucleotide of any one of (a) to (d);

(j) nucleic acid molecules encoding a polypeptide having acetyl-CoAcarboxylase activity, wherein said polypeptide is recognized byantibodies that have been raised against a polypeptide encoded by anucleic acid molecule of any one of (a) to (h);

(k) nucleic acid molecules obtainable by screening an appropriatelibrary under stringent conditions with a probe having the sequence ofthe nucleic acid molecule of any one of (a) to (j), and encoding apolypeptide having an acetyl-CoA carboxylase activity;

(l) nucleic acid molecules whose complementary strand hybridizes understringent conditions with a nucleic acid molecule of any one of (a) to(k), and encoding a polypeptide having acetyl-CoA carboxylase activity.

The terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”,“nucleotide sequence”, “DNA sequence” or “nucleic acid molecule(s)” asused herein refers to a polymeric form of nucleotides of any length,either ribonucleotides or deoxyribonucleotides. This term refers only tothe primary structure of the molecule.

Thus, this term includes double- and single-stranded DNA, and RNA. Italso includes known types of modifications, for example, methylation,“caps” substitution of one or more of the naturally occurringnucleotides with an analog. Preferably, the DNA sequence of theinvention comprises a coding sequence encoding the above-definedpolypeptide.

A “coding sequence” is a nucleotide sequence which is transcribed intomRNA and/or translated into a polypeptide when placed under the controlof appropriate regulatory sequences. The boundaries of the codingsequence are determined by a translation start codon at the 5′-terminusand a translation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to mRNA, cDNA, recombinant nucleotidesequences or genomic DNA, while introns may be present as well undercertain circumstances. SEQ ID:1 depicts the genomic DNA in which theintron sequence is inserted in the coding sequence for acetyl-CoAcarboxylase gene from P. rhodozyma.

In general, the gene consists of several parts which have differentfunctions from each other. In eukaryotes, genes which encode acorresponding protein, are transcribed to premature messenger RNA(pre-mRNA) differing from the genes for ribosomal RNA (rRNA), smallnuclear RNA (snRNA) and transfer RNA (tRNA). Although RNA polymerase II(PolII) plays a central role in this transcription event, PolII can notsolely start transcription without cis element covering an upstreamregion containing a promoter and an upstream activation sequence (UAS),and a trans-acting protein factor. At first, a transcription initiationcomplex which consists of several basic protein components recognize thepromoter sequence in the 5′-adjacent region of the gene to be expressed.In this event, some additional participants are required in the case ofthe gene which is expressed under some specific regulation, such as aheat shock response, or adaptation to a nutrition starvation, and so on.In such a case, a UAS is required to exist in the 5′-untranslatedupstream region around the promoter sequence, and some positive ornegative regulator proteins recognize and bind to the UAS. The strengthof the binding of transcription initiation complex to the promotersequence is affected by such a binding of the transacting factor aroundthe promoter, and this enables the regulation of transcription activity.

After the activation of a transcription initiation complex by thephosphorylation, a transcription initiation complex initiatestranscription from the transcription start site. Some parts of thetranscription initiation complex are detached as an elongation complexfrom the promoter region to the 3′ direction of the gene (this step iscalled as a promoter clearance event) and the elongation complexcontinues the transcription until it reaches to a termination sequencethat is located in the-3′-adjacent downstream region of the gene.Pre-mRNA thus generated is modified in nucleus by the addition of capstructure at the cap site which almost corresponds to the transcriptionstart site, and by the addition of polyA stretches at the polyA signalwhich is located at the 3′-adjacent downstream region. Next, intronstructures are removed from the coding region and exon parts arecombined to yield an open reading frame whose sequence corresponds tothe primary amino acid sequence of a corresponding protein. Thismodification in which a mature mRNA is generated is necessary for astable gene expression. cDNA in general terms corresponds to the DNAsequence which is reverse-transcribed from this mature mRNA sequence. Itcan be synthesized by the reverse transcriptase derived from viralspecies by using a mature mRNA as a template, experimentally.

To express a gene which was derived from eukaryote, a procedure in whichcDNA is cloned into an expression vector for E. coli is often used. Thisresults from the fact that a specificity of intron structure variesamong the organisms and an inability to recognize the intron sequencefrom other species. In fact, prokaryote has no intron structure in itsown genetic background. Even in yeast, the genetic background isdifferent between Ascomycetes to which Saccharomyces cerevisiae belongsand Basidiomycetes to which P. rhodozyma belongs, e.g. the intronstructure of the actin gene from P. rhodozyma cannot be recognized norspliced by the ascomycetous yeast, S. cerevisiae.

Intron structures of some kinds of the genes appear to be involved inthe regulation of the expression of their genes. It might be importantto use a genomic fragment which has its introns in a case ofself-cloning of the gene of a interest whose intron structure involvessuch a regulation of its own gene expression.

To apply a genetic engineering method for a strain improvement study, itis necessary to study its genetic mechanism in the event such astranscription and translation. It is important to determine a geneticsequence such as its UAS, promoter, intron structure and terminator tostudy the genetic mechanism.

According to this invention, the gene encoding the acetyl-CoAcarboxylase (ACC) gene from P. rhodozyma including its 5′- and3′-adjacent regions as well as its intron structure was determined.

The invention further encompasses polynucleotides that differ from oneof the nucleotide sequences shown in SEQ ID NO:2 (and portions thereof)due to degeneracy of the genetic code and also encode an acetyl-CoAcarboxylase as that encoded by the nucleotide sequences shown in SEQ IDNO:2. Further the polynucleotide of the invention has a nucleotidesequence encoding a protein having an amino acid sequence shown in SEQID NO:3. In a still further embodiment, the polynucleotide of theinvention encodes a full length P. rhodozyma protein which issubstantially homologous to an amino acid sequence of SEQ ID NO:3.

In addition, it will be appreciated by those skilled in the art that DNAsequence polymorphism that lead to changes in the amino acid sequencesmay exist within a population (e.g., the P. rhodozyma population). Suchgenetic polymorphism in the acetyl-CoA carboxylase gene may exist amongindividuals within a population due to natural variation.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding an acetyl-CoAcarboxylase, preferably an acetyl-CoA carboxylase from P. rhodozyma.

Such natural variations can typically result in 1-5% variance in thenucleotide sequence of the acetyl-CoA carboxylase gene. Any and all suchnucleotide variations and resulting amino acid polymorphism inacetyl-CoA carboxylase that are the result of natural variation and thatdo not alter the functional activity of acetyl-CoA carboxylase areintended to be within the scope of the invention.

Polynucleotides corresponding to natural variants and non-P. rhodozymahomologues of the acetyl-CoA carboxylase cDNA of the invention can beisolated based on their homology to P. rhodozyma acetyl-CoA carboxylasepolynucleotides disclosed herein using the polynucleotide of theinvention, or a portion thereof, as a hybridization probe according tostandard hybridization techniques under stringent hybridizationconditions. Accordingly, in another embodiment, a polynucleotide of theinvention is at least 15 nucleotides in length. Preferably it hybridizesunder stringent conditions to the nucleic acid molecule comprising anucleotide sequence of the polynucleotide of the present invention, e.g.SEQ ID NO:2. In other embodiments, the nucleic acid is at least 20, 30,50, 100, 250 or more nucleotides in length. The term “hybridizes understringent conditions” is defined above and is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% identical to each other typically remainhybridized to each other. Preferably, the conditions are such thatsequences at least about 65% or 70%, more preferably at least about 75%or 80%, and even more preferably at least about 85%, 90% or 95% or moreidentical to each other typically remain hybridized to each other.Preferably, polynucleotide of the invention that hybridizes understringent conditions to a sequence of SEQ ID NO:2 corresponds to anaturally occurring nucleic acid molecule.

In the present invention, the polynucleotide sequence includes SEQ IDNO:2 and fragments thereof having polynucleotide sequences whichhybridize to SEQ ID NO:2 under stringent conditions which are sufficientto identify specific binding to SEQ ID NO:2. For example, anycombination of the following hybridization and wash conditions may beused to achieve the required specific binding:

High Stringent Hybridization: 6×SSC, 0.5% SDS, 100 μg/ml denaturedsalmon sperm DNA, 50% formamide, incubate overnight with gentle rockingat 42° C.

High Stringent Wash: 1 wash in 2×SSC, 0.5% SDS at room temperature for15 minutes, followed by another wash in 0.1×SSC, 0.5% SDS at roomtemperature for 15 minutes.

Low Stringent Hybridization: 6×SSC, 0.5% SDS, 100 μg/ml denatured salmonsperm DNA, 50% formamide, incubate overnight with gentle rocking at 37°C.

Low Stringent Wash: 1 wash in 0.1×SSC, 0.5% SDS at room temperature for15 minutes.

Moderately stringent conditions may be obtained by varying thetemperature at which the hybridization reaction occurs and/or the washconditions as set forth above. In the present invention, it is preferredto use high stringent hybridization and wash conditions to define theantisense activity against acetyl-CoA carboxylase gene from P.rhodozyma.

The term “homology” means that the respective nucleic acid molecules orencoded proteins are functionally and/or structurally equivalent. Thenucleic acid molecules that are homologous to the nucleic acid moleculesdescribed above and that are derivatives of said nucleic acid moleculesare, for example, variations of said nucleic acid molecules whichrepresent modifications having the same biological function, inparticular encoding proteins with the same or substantially the samebiological function. They may be naturally occuring variations, such assequences from other plant varieties or species, or mutations. Thesemutations may occur naturally or may be obtained by mutagenesistechniques. The allelic variations may be naturally occurring allelicvariants as well as synthetically produced or genetically engineeredvariants. Structural equivalents can, for example, be identified bytesting the binding of said polypeptides to antibodies. Structuralequivalents have similar immunological characteristics, e.g. comprisesimilar epitopes.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein). Preferably, the polynucleotideencodes a natural P. rhodozyma acetyl-CoA carboxylase.

In addition to naturally-occurring variants of the acetyl-CoAcarboxylase sequence that may exist in the population, the skilledartisan will further appreciate that changes can be introduced bymutation into a nucleotide sequence of the polynucleotide encodingacetyl-CoA carboxylase, thereby leading to changes in the amino acidsequence of the encoded acetyl-CoA carboxylase, without altering thefunctional ability of the acetyl-CoA carboxylase. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in a sequence of thepolynucleotide encoding acetyl-CoA carboxylase, e.g. SEQ ID NO:2. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of one of the acetyl-CoA carboxylase withoutaltering the activity of said acetyl-CoA carboxylase, whereas an“essential” amino acid residue is required for acetyl-CoA carboxylaseactivity. Other amino acid residues, however, (e.g., those that are notconserved or only semi-conserved in the domain having acetyl-CoAcarboxylase activity) may not be essential for activity and thus arelikely to be amenable to alteration without altering acetyl-CoAcarboxylase activity.

Accordingly, the invention relates to polynucleotides encodingacetyl-CoA carboxylase that contain changes in amino acid residues thatare not essential for acetyl-CoA carboxylase activity. Such acetyl-CoAcarboxylase differs in amino acid sequence from a sequence contained inSEQ ID NO:3 yet retain the acetyl-CoA carboxylase activity describedherein. The polynucleotide can comprise a nucleotide sequence encoding apolypeptide, wherein the polypeptide comprises an amino acid sequence atleast about 60% identical to an amino acid sequence of SEQ ID NO:3 andhas acetyl-CoA carboxylase activity. Preferably, the protein encoded bythe nucleic acid molecule is at least about 60-65% identical to thesequence in SEQ ID NO:3, more preferably at least about 60-70% identicalto one of the sequences in SEQ ID NO:3, even more preferably at leastabout 70-80%, 80-90%, 90-95% homologous to the sequence in SEQ ID NO:3,and most preferably at least about 96%, 97%, 98%, or 99% identical tothe sequence in SEQ ID NO:3.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequence of SEQ ID NO:3 and a mutant form thereof) or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of one protein or nucleicacid for optimal alignment with the other protein or nucleic acid). Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in onesequence (e.g., one of the sequences of SEQ ID NO:2 or 3) is occupied bythe same amino acid residue or nucleotide as the corresponding positionin the other sequence (e.g., a mutant form of the sequence selected),then the molecules are homologous at that position (i.e., as used hereinamino acid or nucleic acid “homology” is equivalent to amino acid ornucleic acid “identity”). The percent homology between the two sequencesis a function of the number of identical positions shared by thesequences (i.e., % homology=numbers of identical positions/total numbersof positions×100). The homology can be determined by computer programsas Blast 2.0 [Altschul, Nuc. Acid. Res., 25:3389-3402 (1997)]. In thisinvention, GENETYX-SV/RC software (Software Development Co., Ltd.,Tokyo, Japan) is used by using its default algorithm as such homologyanalysis software. This software uses the Lipman-Pearson method for itsanalytic algorithm.

A nucleic acid molecule encoding an acetyl-CoA carboxylase homologous toa protein with an amino acid sequence of SEQ ID NO:3 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto a nucleotide sequence of the polynucleotide of the presentinvention, in particular of SEQ ID NO:2 such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced into the sequences of, e.g., SEQ IDNO:2 by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in anacetyl-CoA carboxylase is preferably replaced with another amino acidresidue from the same family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of an acetyl-CoAcarboxylase coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for an acetyl-CoA carboxylase activitydescribed herein to identify mutants that retain acetyl-CoA carboxylaseactivity. Following mutagenesis of one of the sequences of SEQ ID NO:2,the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined using, for example, assays describedherein.

A polynucleotide of the present invention, e.g., a nucleic acid moleculehaving a nucleotide sequence of SEQ ID NO:2, or a portion thereof, canbe isolated using standard molecular biology techniques and the sequenceinformation provided herein. For example, acetyl-CoA carboxylase cDNAcan be isolated from a library using all or portion of one of thesequences of the polynucleotide of the present invention as ahybridization probe and standard hybridization techniques. Moreover, apolynucleotide encompassing all or a portion of one of the sequences ofthe polynucleotide of the present invention can be isolated by thepolymerase chain reaction using oligonucleotide primers designed basedupon this sequence (e.g., a nucleic acid molecule encompassing all or aportion of one of the sequences of polynucleotide of the presentinvention can be isolated by the polymerase chain reaction usingoligonucleotide primers, e.g. of SEQ ID NO:4, 5, or 6, designed basedupon this same sequence of polynucleotide of the present invention. Forexample, mRNA can be isolated from cells, e.g. Phaffia (e.g., by theguanidinium-thiocyanate extraction procedure of Chirgwin et al. and cDNAcan be prepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase or AMV reverse transcriptase available from Promega(Madison, USA)). Synthetic oligonucleotide primers for polymerase chainreaction amplification can be designed based upon one of the nucleotidesequences shown in SEQ ID NO:2. A polynucleotide of the invention can beamplified using cDNA or, alternatively, genomic DNA, as a template andappropriate oligonucleotide primers according to standard PCRamplification techniques. The polynucleotide so amplified can be clonedinto an appropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to an acetyl-CoA carboxylasenucleotide sequence can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

The terms “fragment”, “fragment of a sequence” or “part of a sequence”means a truncated sequence of the original sequence referred to. Thetruncated sequence (nucleic acid or protein sequence) can vary widely inlength; the minimum size being a sequence of sufficient size to providea sequence with at least a comparable function and/or activity of theoriginal sequence referred to, while the maximum size is not critical.In some applications, the maximum size usually is not substantiallygreater than that required to provide the desired activity and/orfunction(s) of the original sequence.

Typically, the truncated amino acid sequence will range from about 5 toabout 60 amino acids in length. More typically, however, the sequencewill be a maximum of about 50 amino acids in length, preferably amaximum of about 30 amino acids. It is usually desirable to selectsequences of at least about 10, 12 or 15 amino acids, up to maximum ofabout 20 or 25 amino acids.

The term “epitope” relates to specific immunoreactive sites within anantigen, also known as antigenic determinants. These epitopes can be alinear array of monomers in a polymeric composition—such as amino acidsin a protein—or consist of or comprise a more complex secondary ortertiary structure. Those of skill will recognize that all immunogens(i. e., substances capable of eliciting an immune response) areantigens; however, some antigen, such as haptens, are not immunogens butmay be made immunogenic by coupling to a carrier molecule. The term“antigen” includes references to a substance to which an antibody can begenerated and/or to which the antibody is specifically immunoreactive.

The term “one or several amino acids” relates to at least one amino acidbut not more than that number of amino acids which would result in ahomology of below 60% identity. Preferably, the identity is more than70% or 80%, more preferred are 85%, 90% or 95%, even more preferred are96%, 97%, 98%, or 99% identity.

The term “acetyl-CoA carboxylase” or “acetyl-CoA carboxylase activity”relates to enzymatic activities of a polypeptide as described below orwhich can be determined in enzyme assay method. Furthermore,polypeptides that are inactive in an assay herein but are recognized byan antibody specifically binding to acetyl-CoA carboxylase, i.e., havingone or more acetyl-CoA carboxylase epitopes, are also comprised underthe term “acetyl-CoA carboxylase”. In these cases activity refers totheir immunological activity.

The terms “polynucleotide” and “nucleic acid molecule” also relate to“isolated” polynucleotides or nucleic acids molecules. An “isolated”nucleic acid molecule is one which is separated from other nucleic acidmolecules which are present in the natural source of the nucleic acid.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived.

For example, in various embodiments, the PNO polynucleotide can containless than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule ingenomic DNA of the cell from which the nucleic acid is derived (e.g., aPhaffia cell). Moreover, the polynucleotides of the present invention,in particular an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized.

Preferably, the polypeptide of the invention comprises one of thenucleotide sequences shown in SEQ ID NO:2. The sequence of SEQ ID NO:2corresponds to the P. rhodozyma acetyl-CoA carboxylase cDNAs of theinvention.

Further, the polynucleotide of the invention comprises a nucleic acidmolecule which is a complement of one of the nucleotide sequences ofabove mentioned polynucleotides or a portion thereof. A nucleic acidmolecule which is complementary to one of the nucleotide sequences shownin SEQ ID NO:2 is one which is sufficiently complementary to one of thenucleotide sequences shown in SEQ ID NO:2 such that it can hybridize toone of the nucleotide sequences shown in SEQ ID NO:2, thereby forming astable duplex.

The polynucleotide of the invention comprises a nucleotide sequencewhich is at least about 60%, preferably at least about 65-70%, morepreferably at least about 70-80%, 80-90%, or 90-95%, and even morepreferably at least about 95%, 96%, 97%, 98%, 99% or more homologous toa nucleotide sequence shown in SEQ ID NO:2, or a portion thereof Thepolynucleotide of the invention comprises a nucleotide sequence whichhybridizes, e.g., hybridizes under stringent conditions as definedherein, to one of the nucleotide sequences shown in SEQ ID NO:2, or aportion thereof.

Moreover, the polynucleotide of the invention can comprise only aportion of the coding region of one of the sequences in SEQ ID NO:2, forexample a fragment which can be used as a probe or primer or a fragmentencoding a biologically active portion of an acetyl-CoA carboxylase. Thenucleotide sequences determined from the cloning of the acetyl-CoAcarboxylase gene from P. rhodozyma allows for the generation of probesand primers designed for use in identifying and/or cloning acetyl-CoAcarboxylase homologues in other cell types and organisms. Theprobe/primer typically comprises a substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, 15 preferably about 20 or 25, more preferably about 40,50 or 75 consecutive nucleotides of a sense strand of one of thesequences set forth, e.g., in SEQ ID NO: No:2, an anti-sense sequence ofone of the sequences, e.g., set forth in SEQ ID NO:2, or naturallyoccurring mutants thereof. Primers based on a nucleotide of inventioncan be used in PCR reactions to clone acetyl-CoA carboxylase homologues.Probes based on the acetyl-CoA carboxylase nucleotide sequences can beused to detect transcripts or genomic sequences encoding the same orhomologous proteins. The probe can further comprise a label groupattached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a genomic marker test kit for identifying cellswhich express an acetyl-CoA carboxylase, such as by measuring a level ofan acetyl-CoA carboxylase-encoding nucleic acid molecule in a sample ofcells, e.g., detecting acetyl-CoA carboxylase mRNA levels or determiningwhether a genomic acetyl CoA carboxylase gene has been mutated ordeleted.

The polynucleotide of the invention encodes a polypeptide or portionthereof which includes an amino acid sequence which is sufficientlyhomologous to an amino acid sequence of SEQ ID NO:3 such that theprotein or portion thereof maintains an acetyl-CoA carboxylase activity,in particular an acetyl-CoA carboxylase activity as described in theexamples in microorganisms or plants. As used herein, the language“sufficiently homologous” refers to proteins or portions thereof whichhave amino acid sequences which include a minimum number of identical orequivalent (e.g., an amino acid residue which has a similar side chainas an amino acid residue in one of the sequences of the polypeptide ofthe present invention amino acid residues to an amino acid sequence ofSEQ ID NO:3 such that the protein or portion thereof has an acetyl-CoAcarboxylase activity. Examples of an acetyl-CoA carboxylase activity arealso described herein.

The protein is at least about 60-65%, preferably at least about 66-70%,and more preferably at least about 70-80%, 80-90%, 90-95%, and mostpreferably at least about 96%, 97%, 98%, 99% or more homologous to anentire amino acid sequence of SEQ ID NO:3.

Portions of proteins encoded by the acetyl-CoA carboxylasepolynucleotide of the invention are preferably biologically activeportions of one of the acetyl-CoA carboxylase.

As mentioned herein, the term “biologically active portion of acetyl-CoAcarboxylase” is intended to include a portion, e.g., a domain/motif,that has acetyl-CoA carboxylase activity or has an immunologicalactivity such that it is binds to an antibody binding specifically toacetyl-CoA carboxylase. To determine whether an acetyl-CoA carboxylaseor a biologically active portion thereof can participate in themetabolism an assay of enzymatic activity may be performed. Such assaymethods are well known to those skilled in the art, as detailed in theExamples. Additional nucleic acid fragments encoding biologically activeportions of an acetyl-CoA carboxylase can be prepared by isolating aportion of one of the sequences in SEQ ID NO:2, expressing the encodedportion of the acetyl-CoA carboxylase or peptide (e.g., by recombinantexpression in vitro) and assessing the activity of the encoded portionof the acetyl-CoA carboxylase or peptide.

At first, a partial gene fragment was cloned containing a portion of theACC gene by using the degenerate PCR method. Said degenerate PCR is amethod to clone a gene of interest which has high homology of amino acidsequence to the known enzyme from other species which has the same orsimilar function. Degenerate primer, which is used as a primer indegenerate PCR, was designed by a reverse translation of the amino acidsequence to corresponding nucleotides (“degenerated”). In such adegenerate primer, a mixed primer which consists any of A, C, G or T, ora primer containing inosine at an ambiguity code is generally used. Inthis invention, such mixed primers were used for degenerate primers toclone above gene.

An entire gene containing its coding region with its intron as well asits regulation region such as a promoter or a terminator can be clonedfrom a chromosome by screening of a genomic library which is constructedin phage vector or plasmid vector in appropriate host, by using apartial DNA fragment obtained by degenerate PCR as described above as aprobe after it was labeled. Generally, E. coli as a host strain and E.coli vector, a phage vector such as λ phage vector, or a plasmid vectorsuch as pUC vector is often used in the construction of a library and afollowing genetic manipulation such as a sequencing, a restrictiondigestion, a ligation and the like. In this invention, an EcoRI genomiclibrary of P. rhodozyma was constructed in the derivatives of λ vector,λZAPII. An insert size, what length of insert must be cloned, wasdetermined by the Southern blot hybridization for the gene beforeconstruction of a library. In this invention, a DNA used for a probe waslabeled with digoxigenin (DIG), a steroid hapten instead of conventional³²P label, following the protocol which was prepared by the supplier(Boehringer-Mannheim, Mannheim, Germany). A genomic library constructedfrom the chromosome of P. rhodozyma was screened by using a DIG-labeledDNA fragment which had a portion of a gene of interest as a probe.Hybridized plaques were picked up and used for further study. WhenλZAPII (insert size was below 9 kb) was used in the construction of thegenomic library, in vivo excision protocol was conveniently used for thesucceeding step of the cloning into the plasmid vector by using aderivative of single stranded M13 phage, Ex assist phage (Stratagene, LaJolla, USA). A plasmid DNA thus obtained was examined for sequencing.

In this invention, we used the automated fluorescent DNA sequencer,ALFred system (Pharmacia, Uppsala, Sweden) using an autocycle sequencingprotocol in which the Taq DNA polymerase is employed in most cases ofsequencing.

After the determination of the genomic sequence, a sequence of a codingregion was used for a cloning of cDNA of corresponding gene. The PCRmethod was also exploited to clone cDNA fragment. The PCR primers whosesequences were identical to the sequence at the 5′- and 3′-end of theopen reading frame (ORF) were synthesized with an addition of anappropriate restriction site, and PCR was performed by using those PCRprimers. In this invention, a cDNA pool was used as a template in thisPCR cloning of cDNA. The said cDNA pool consists of various cDNA specieswhich were synthesized in vitro by the viral reverse transcriptase andTaq polymerase (CapFinder Kit manufactured by Clontech, Palo Alto,U.S.A.) by using the mRNA obtained from P. rhodozyma as a template. cDNAof interest thus obtained was confirmed in its sequence. Furthermore,cDNA thus obtained was used for a confirmation of its enzyme activityafter the cloning of the cDNA fragment into an expression vector whichfunctions in E. coli under the strong promoter activity such as the lacor T7 expression system.

In another embodiment, the present invention relates to a method formaking a recombinant vector comprising inserting a polynucleotide of theinvention into a vector.

Further, the present invention relates to a recombinant vectorcontaining the polynucleotide of the invention or produced by saidmethod of the invention.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting a polynucleotide to which it has been linked.One type of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments can be ligated.Another type of vector is a viral vector, wherein additional DNA or PNAsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The present invention also relates to cosmids, viruses, bacteriophagesand other vectors used conventionally in genetic engineering thatcontain a nucleic acid molecule according to the invention. Methodswhich are well known to those skilled in the art can be used toconstruct various plasmids and vectors. Alternatively, the nucleic acidmolecules and vectors of the invention can be reconstituted intoliposomes for delivery to target cells.

The present invention further relates to a vector in which thepolynucleotide of the present invention is operatively linked toexpression control sequences allowing expression in prokaryotic oreukaryotic host cells. The nature of such control sequences differsdepending upon the host organism. In prokaryotes, control sequencesgenerally include promoter, ribosomal binding site, and terminators. Ineukaryotes, generally control sequences include promoters, terminatorsand, in some instances, enhancers, transactivators; or transcriptionfactors.

The term “control sequence” is intended to include, at a minimum,components the presence of which are necessary for expression, and mayalso include additional advantageous components.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences. In case the control sequence is a promoter, it is obvious fora skilled person that double-stranded nucleic acid is used.

Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in many types of host cell and those whichdirect expression of the nucleotide sequence only in certain host cellsor under certain conditions. It will be appreciated by those skilled inthe art that the design of the expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of protein desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby produce proteinsor peptides, including fusion proteins or peptides, encoded bypolynucleotides as described herein.

The recombinant expression vectors of the invention can be designed forexpression of acetyl-CoA carboxylase in prokaryotic or eukaryotic cells.For example, genes encoding the polynucleotide of the invention can beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors), yeast and other fungal cells, algae,ciliates of the types: Holotrichia, Peritrichia, Spirotrichia, Suctoria,Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyStylonychia lemnae with vectors following, a transformation method asdescribed in WO9801572 and multicellular plant cells. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve three purposes: 1) to increase expression of recombinant protein;2) to increase the solubility of the recombinant protein; and 3) to aidin the purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc.),pMAL (New England Biolabs, Beverly, USA) and pRIT5 (Pharmacia,Piscataway, USA) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein. In one embodiment, the coding sequence of the polypeptideencoded by the polynucleotide of the present invention is cloned into apGEX expression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin, e.g. recombinantacetyl-CoA carboxylase unfused to GST can be recovered by cleavage ofthe fusion protein with thrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc and pET 11d. Target gene expression from the pTrc vectorrelies on host RNA polymerase transcription from a hybrid trp-lac fusionpromoter. Target gene expression from the pET 11d vector relies ontranscription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gnl). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident Xprophage harboring a T7 gnl gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression is to expressthe protein in host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. Another strategy is toalter the nucleic acid sequence of the nudeic acid to be inserted intoan expression vector so that the individual codons for each amino acidare those preferentially utilized in the batterium chosen forexpression, such as E. coli. Such alteration of nucleic acid sequencesof the invention can be carried out by standard DNA synthesistechniques.

Further, the acetyl-CoA carboxylase vector can be a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1, pMFa, pJRY88, and pYES2 (Invitrogen, San Diego, USA).Vectors and methods for the construction of vectors appropriate for usein other fungi, such as the filamentous fungi, are known to the skilledartisan.

Alternatively, the polynucleotide of the invention can be introduced ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series and the pVL series.

Alternatively, the polynucleotide of the invention is introduced inmammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 and pMT2PC. When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40.

The recombinant mammalian expression vector can be capable of directingexpression of the nucleic acid preferentially in a particular cell type(e.g., tissue-specific regulatory elements are used to express thenucleic acid). Tissue-specific regulatory elements are known in the art.Non-limiting examples of suitable tissue-specific promoters include thealbumin promoter (liver-specific), lymphoid-specific promoters, inparticular promoters of T cell receptors and immunoglobulins,neuron-specific promoters (e.g., the neurofilament promoter),pancreas-specific promoters, and mammary gland-specific promoters (e.g.,milk whey promoter; U.S. Pat. No. 4,873, 316 and EP 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters and the fetoprotein promoter.

Thus expressed ACC gene can be verified for its activity, e.g., by anenzyme assay method. Some experimental protocols are described in theliterature. The following is the one of the methods which is used forthe determination of acetyl-CoA carboxylase activity: Assays areperformed by measuring the loss in acetyl-CoA and/or the production ofmalonyl-CoA at 5 min intervals for 20 min, using reverse phase HPLC. Therate of conversion of acetyl-CoA to malonyl-CoA is found to be linearfor 20 min, and velocities are calculated by linear regression analysisof the malonyl-CoA concentration with respect to time. The reactionmixture contained 50 μM Tris, pH 7.5, 6 μM acetyl-CoA, 2 mM ATP, 7 mMKHCO₃, 8 mM MgCl₂, 1 mM dithiothreitol, and 1 mg/ml bovine serumalbumin. Enzyme is preincubated (30 min, 25° C.) with bovine serumalbumin (2 mg/ml) and potassium citrate (10 mM). Reactions are initiatedby transferring 50 μl of preincubated enzyme to the reaction mixture(final volume 200 μl) and incubated for 5-20 min at 25° C. Reactions areterminated by addition of 50 μl 10% perchloric acid. Followingtermination of the reaction, the samples are centrifuged (3 min,10,000×g) and analyzed by HPLC. A mobile phase of 10 mM KH₂PO₄, pH 6.7(solvent A), and MeOH (solvent B) is used. The flow rate is 1.0 ml/min,and the gradient is as follows: hold at 100% solvent A for 1 minfollowed by a linear gradient to 30% solvent B over the next 5 min, thenhold at 30% solvent B for 5 min. Using this method the retention timeswere 7.5 and 9.0 min for malonyl-CoA and acetyl-CoA, respectively. Whenan expression vector for S. cerevisiae is used, a complementationanalysis can be conveniently exploited by using conditional acetyl-CoAcarboxylase null mutant strain derived from S. cerevisiae as a hoststrain for its confirmation of activity.

Succeeding to the confirmation of the enzyme activity, an expressedprotein would be purified and used for raising the antibody against thepurified enzyme. Antibody thus prepared would be used for acharacterization of the expression of the corresponding enzyme in astrain improvement study, an optimization study of the culturecondition, and the like.

In a further embodiment, the present invention relates to an antibodythat binds specifically to the polypeptide of the present invention orparts, i.e. specific fragments or epitopes of such a protein.

The antibodies of the invention can be used to identify and isolateother acetyl-CoA carboxylase and genes. These antibodies can bemonoclonal antibodies, polyclonal antibodies or synthetic antibodies aswell as fragments of antibodies, such as Fab, Fv or scFv fragments etc.Monoclonal antibodies can be prepared, for example, by the techniques asoriginally described by Kohler and Milstein, which comprise the fusionof mouse myeloma cells to spleen cells derived from immunized mammals.

Furthermore, antibodies or fragments thereof to the aforementionedpeptides can be obtained by using methods known to the skilled person.These antibodies can be used, for example, for the immunoprecipitationand immunolocalization of proteins according to the invention as well asfor the monitoring of the synthesis of such proteins, for example, inrecombinant organisms, and for the identification of compoundsinteracting with the protein according to the invention. For example,surface plasmon resonance as employed in the BIAcore system can be usedto increase the efficiency of phage antibodies selections, yielding ahigh increment of affinity from a single library of phage antibodieswhich bind to an epitope of the protein of the invention. In many cases,the binding phenomenon of antibodies to antigens is equivalent to otherligand/anti-ligand binding.

In this invention, the gene fragment for acetyl-CoA carboxylase wascloned from P. rhodozyma with a purpose to decrease its expression levelin P. rhodozyma by genetic method using the cloned gene fragment.

To decrease a gene expression with genetic methods, some strategies canbe employed, one of which is a gene-disruption method. In this method, apartial fragment of the objective gene to be disrupted is ligated to adrug resistant cassette on the integration vector which can notreplicate in the host organism. A drug resistance gene which encodes theenzyme that enables the host to survive in the presence of a toxicantibiotic is often used for the selectable marker. G418 resistance geneharbored in pGB-Ph9 (Wery et al. (Gene, 184, 89-97, 1997)) is an exampleof a drug resistance gene which functions in P. rhodozyma. Nutritioncomplementation marker can be also used in the host which has anappropriate auxotrophy marker. P. rhodozyma ATCC24221 strain thatrequires cytidine for its growth is one example of the auxotroph. Byusing CTP synthetase as donor DNA for ATCC24221, a host vector systemusing a nutrition complementation can be established.

After the transformation of the host organisms and recombination betweenthe objective gene fragment on the vector and its corresponding genefragment on the chromosome of the host organisms, the integration vectoris integrated onto the host chromosome by single cross recombination. Asa result of this recombination, the drug resistant cassette would beinserted in the objective gene whose translated product is onlysynthesized in its truncated form which does not have its enzymaticfunction. In a similar manner, two parts of the objective gene were alsoused for gene disruption study in which the drug resistant gene can beinserted between such two partial fragments of the objective genes onthe integration vector. In the case of this type of vector, doublerecombination event between the gene fragments harbored on theintegration vector and the corresponding gene fragments on thechromosome of the host are expected. Although frequency of this doublecrossing-over recombination is lower than single cross recombination,null, phenotype of the objective gene by the double cross recombinationis more stable than by the single cross recombination.

On the other hand, this strategy has difficulty in the case of the genewhose function is essential and disruption is lethal for the hostorganism such as acetyl-CoA carboxylase gene. The function of acetyl-CoAcarboxylase is indispensable for the host survival other than thebiosynthesis of fatty acid. From such a viewpoint, it seemed to bedifficult to construct the acetyl-CoA carboxylase disruptant from P.rhodozyma by this gene disruption method.

In such a case, other strategies can be applied to decrease (not todisrupt) a gene expression, one of which is a conventional mutagenesisto screen the mutant whose expression for acetyl-CoA carboxylase isdecreased. In this method, an appropriate recombinant in which anappropriate reporter gene is fused to the promoter region of acetyl-CoAcarboxylase gene from the host organism is mutated and mutants whichshow a weaker activity of reporter gene product can be screened. In suchmutants, it is expected that their expression of acetyl-CoA carboxylaseactivity decreased by the mutation lying in the promoter region ofreporter gene or trans-acting region which might affect the expressionof acetyl-CoA carboxylase gene other than the mutation lying in thepromoter gene itself In the case of mutation occurring at the promoterregion of the reporter fusion, such mutation can be isolated by thesequence of the corresponding region. Thus isolated mutation can beintroduced in a variety of carotenoids, especially astaxanthin producingmutants derived from P. rhodozyma by a recombination between theoriginal promoter for acetyl-CoA carboxylase gene on the chromosome andthe mutated promoter fragment. To exclude mutations occurring at atrans-acting region, a mutation can also be induced by an in vitromutagenesis of a cis element in the promoter region. In this approach, agene cassette, containing a reporter gene which is fused to a promoterregion derived from a gene of interest at its 5′-end and a terminatorregion from a gene of interest at its 3′-end, is mutagenized and thenintroduced into P. rhodozyma. By detecting the difference of theactivity of the reporter gene, an effective mutation can be screened.Such a mutation can be introduced in the sequence of the native promoterregion on the chromosome by the same method as the case of an in vivomutation approach. But, these methods have some drawbacks to have sometime-consuming process.

Another strategy to decrease a gene expression is an antisense method.This method is frequently applied to decrease the gene expression evenwhen teleomorphic organisms such as P. rhodozyma are used as hostorganisms, to which the mutation and gene disruption method is usuallydifficult to be applied. The anti-sense method is a method to decreasean expression of gene of interest by introducing an artificial genefragment, whose sequence is complementary to cDNA fragment of the geneof interest. Such an anti-sense gene fragment would form a complex witha mature mRNA fragment of the objective gene in vivo and inhibit anefficient translation from mRNA, as a consequence.

An “antisense” nucleic acid molecule comprises a nucleotide sequencewhich is complementary to a “sense” nucleic acid molecule encoding aprotein, e. g., complementary to the coding strand of a double-strandedcDNA molecule or complementary to a mRNA sequence. Accordingly, anantisense nucleic acid molecule can hydrogen bond to a sense nucleicacid molecule. The antisense nucleic acid molecule can be complementaryto an entire acetyl-CoA carboxylase-coding strand, or to only a portionthereof. Accordingly, an antisense nucleic acid molecule can beantisense to a “coding region” of the coding strand of a nucleotidesequence encoding an acetyl-CoA carboxylase. The term “coding region”refers to the region of the nucleotide sequence comprising codons whichare translated into amino acid residues. Further, the antisense nucleicacid molecule is antisense to a “noncoding region” of the coding strandof a nucleotide sequence encoding acetyl-CoA carboxylase. The term“noncoding region” refers to 5′ and 3′ sequences which flank the codingregion that are not translated into a polypeptide (i.e., also referredto as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding acetyl-CoA carboxylasedisclosed herein, antisense nucleic acid molecules of the invention canbe designed according to the rules of Watson and Crick base pairing. Theantisense nucleic acid molecule can be complementary to the entirecoding region of acetyl-CoA carboxylase mRNA, but can also be anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of acetyl-CoA carboxylase mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of acetyl-CoA carboxylase mRNA. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 nucleotides in length. An antisense nucleic acid moleculeof the invention can be constructed using chemical synthesis andenzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid molecule (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the anti-sense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethyl-aminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine; 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which apolynucleotide has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted polynucleotide will be of an antisenseorientation to a target polynucleotide of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an acetyl-CoAcarboxylase to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The anti-sense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic includingplant promoters are preferred.

The antisense nucleic acid molecule of the invention may, e.g., be anα-anomeric nucleic acid molecule. An α-anomeric nucleic acid moleculeforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other.The antisense nucleic acid molecule can also comprise a2α-o-methylribonucleotide or a chimeric RNA-DNA analogue.

Further the antisense nucleic acid molecule of the invention can be aribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as a mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes) can be used to catalyticallycleave acetyl-CoA carboxylase mRNA transcripts to thereby inhibittranslation of mRNA. A ribozyme having specificity for an acetyl-CoAcarboxylase-encoding nucleic acid molecule can be designed based uponthe nucleotide sequence of an acetyl-CoA carboxylase cDNA disclosedherein or on the basis of a heterologous sequence to be isolatedaccording to methods taught in this invention. For example, a derivativeof a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in an encoding mRNA (see, e.g., U.S. Pat. No. 4,987,071and U.S. Pat. No. 5,116,742). Alternatively, acetyl-CoA carboxylase mRNAcan be used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules.

The application of the antisense method to construct a carotenoidoverproducing strain from P. rhodozyma is disclosed in EP 1,158,051.

In one embodiment the present invention relates to a method of making arecombinant host cell comprising introducing the vector or thepolynucleotide of the present invention into a host cell.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection”, conjugation andtransduction are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer, or electroporation. Suitable methods fortransforming or transfecting host cells including plant cells are knownto the skilled artisan.

For stable transfection of mammalian cells, only a small fraction ofcells may integrate the foreign DNA into their genome, depending uponthe expression vector and transfection technique used. In order toidentify and select these integrants, a gene that encodes a selectablemarker (e.g., resistance to antibiotics) is generally introduced intothe host cells along with the gene of interest. Preferred selectablemarkers include those which confer resistance to drugs, such as G418,hygromycin and methotrexate. Nucleic acid encoding a selectable markercan be introduced into a host cell on the same vector as that encodingthe polypeptide of the present invention or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by, for example, drug selection (e.g;, cells thathave incorporated the selectable marker gene will survive, while theother cells die).

To create a homologous recombinant microorganism, a vector is preparedwhich contains at least a portion of the polynucleotide of the presentinvention into which a deletion, addition or substitution has beenintroduced to thereby alter, e.g., functionally disrupt, the acetyl-CoAcarboxylase gene. Preferably, this acetyl-CoA carboxylase gene is a P.rhodozyma acetyl-CoA carboxylase gene, but it can be a homologue from arelated or different source. Alternatively, the vector can be designedsuch that, upon homologous recombination, the endogenous acetyl-CoAcarboxylase gene is mutated or otherwise altered but still encodes afunctional protein (e.g., the upstream regulatory region can be alteredto thereby alter the expression of the endogenous acetyl-CoAcarboxylase). To create a point mutation via homologous recombinationalso DNA-RNA hybrids can be used known as chimeraplasty known fromCole-Strauss et al., Nucl. Aci. Res., 27, 5, 1323-1330, 1999 and Kmiec,Gene therapy., American Scientist. 87, 3, 240-247. 1999.

The vector is introduced into a cell and cells in which the introducedpolynucleotide gene has homologously recombined with the endogenousacetyl-CoA carboxylase gene are selected, using art-known techniques.

Further host cells can be produced which contain selection systems whichallow for regulated expression of the introduced gene. For example,inclusion of the polynucleotide of the invention on a vector placing itunder control of the lac operon permits expression of the polynucleotideonly in the presence of IPTG. Such regulatory systems are well known inthe art.

Preferably, the introduced nucleic acid molecule is foreign to the hostcell.

By “foreign” it is meant that the nucleic acid molecule is eitherheterologous with, respect to the host cell, this means derived from acell or organism with a different genomic background, or is homologouswith respect to the host cell but located in a different genomicenvironment than the naturally occurring counterpart of said nucleicacid molecule. This means that, if the nucleic acid molecule ishomologous with respect to the host cell, it is not located in itsnatural location in the genome of said host cell, in particular it issurrounded by different genes. In this case the nucleic acid moleculemay be either under the control of its own promoter or under the controlof a heterologous promoter. The vector or nucleic acid moleculeaccording to the invention which is present in the host cell may eitherbe integrated into the genome of the host cell or it may be maintainedin some form extrachromosomally. In this respect, it is also to beunderstood that the nucleic acid molecule of the invention can be usedto restore or ceate a mutant gene via homologous recombination.

Accordingly, in another embodiment the present invention relates to ahost cell genetically engineered with the polynucleotide of theinvention or the vector of the invention.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

For example, a polynucleotide of the present invention can be introducedin bacterial cells as well as insect cells, fungal cells or mammaliancells (such as Chinese hamster ovary cells (CHO) or COS cells), algae,ciliates, plant cells, fungi or other microorganims like E. coli. Othersuitable host cells are known to those skilled in the art. Preferred areE. coli, baculovirus, Agrobacterium or fungal cells are, for example,those of the genus Saccharomyces, e.g. those of the species S.cerevisiae or P. rhodozyna (Xanthophylomyces dendrorhous).

In addition, in one embodiment, the present invention relates to amethod for the production of fungal transformants comprising theintroduction of the polynucleotide or the vector of the presentinvention into the genome of said fungal cell.

For the expression of the nucleic acid molecules according to theinvention in sense or antisense orientation in plant cells, themolecules are placed under the control of regulatory elements whichensure the expression in fungal cells. These regulatory elements may beheterologous or homologous with respect to the nucleic acid molecule tobe expressed as well with respect to the fungal species to betransformed.

In general, such regulatory elements comprise a promoter active infungal cells. To obtain constitutive expression in fungal cells,preferably constitutive promoters are used, e.g., theglyceraldehyde-3-dehydrogenase promoter derived from P. rhodozyma (WO97/23,633). Inducible promoters may be used in order to be able toexactly control expression. An example for inducible promoters is thepromoter of genes encoding heat shock proteins. Also an amylase genepromoter which is a candidate for such inducible promoters has beendescribed (EP 1,035,206). The regulatory elements may further comprisetranscriptional and/or translational enhancers functional in fungalcells. Furthermore, the regulatory elements may include transcriptiontermination signals, such as a poly-A signal, which lead to the additionof a poly A tail to the transcript which may improve its stability.

Methods for the introduction of foreign DNA into fungal cells are alsowell known in the art. These include, for example, transformation withthe LiCl method, the fusion of protoplasts, electroporation, biolisticmethods like particle bombardment other methods known in the art.Methods for the transformation using biolistic methods are well known tothe person skilled in the art.

The term “transformation” as used herein, refers to the transfer of anexogenous polynucleotide into a host cell, irrespective of the methodused for the transfer. The polynucleotide may be transiently or stablyintroduced into the host cell and may be maintained non-integrated, forexample, as a plasmid or as chimeric links, or alternatively, may beintegrated into the host genome.

In general, the fungi which can be modified according to the inventionand which either show overexpression of a protein according to theinvention or a reduction of the synthesis of such a protein can bederived from any desired fungal species.

Further, in one embodiment, the present invention relates to a fungalcell comprising the polynucleotide the vector or obtainable by themethod of the present invention.

Thus, the present invention relates also to transgenic fungal cellswhich contain (preferably stably integrated into the genome) apolynucleotide according to the invention linked to regulatory elementswhich allow expression of the polynucleotide in fungal cells and whereinthe polynucleotide is foreign to the transformed fungal cell. For themeaning of foreign; see supra.

Thus, the present invention also relates to transformed fungal cellsaccording to the invention.

Accordingly, due to the altered expression of acetyl-CoA carboxylase,cells metabolic pathways are modulated in yield production, and/orefficiency of production.

The terms “production” or “productivity” are art-recognized and includethe concentration of the fermentation product (for example fatty acids,carotenoids, (poly)saccharides, lipids, vitamins, isoprenoids, waxesters, and/or polymers like polyhydroxyalkanoates and/or its metabolismproducts or further desired fine chemical as mentioned herein) formedwithin a given time and a given fermentation volume (e.g., kg productper hour per liter).

The term “efficiency” of production includes the time required for aparticular level of production to be achieved (for example, how long ittakes for the cell to attain a particular rate of output of a saidaltered yield, in particular, into carotenoids, (poly)saccharides,lipids, vitamins, isoprenoids etc.).

The term “yield” or “product/carbon yield” is art-recognized andincludes the efficiency of the conversion of the carbon source into theproduct (i.e. acetyl CoA, fatty acids, vitamins, carotenoids,isoprenoids, lipids etc. and/or further compounds as defined above andwhich biosynthesis is based on said products). This is generally writtenas, for example, kg product per kg carbon source. By increasing theyield or production of the compound, the quantity of recoveredmolecules, or of useful recovered molecules of that compound in a givenamount of culture over a given amount of time is increased.D-galactosylqueosine,

The terms “biosynthesis” (which is used synonymously for “synthesis” of“biological production” in cells, tissues plants, etc.) or a“biosynthetic pathway” are art-recognized and include the synthesis of acompound, preferably an organic compound, by a cell from intermediatecompounds in what may be a multistep and highly regulated process.

The language “metabolism” is art-recognized and includes the totality ofthe biochemical reactions that take place in an organism. The metabolismof a particular compound, then, (e.g., the metabolism of acetyl CoA, afatty acid, hexose, isoprenoid, vitamin, carotenoid, lipid etc.)comprises the overall biosynthetic, modification, and degradationpathways in the cell related to this compound.

Such a genetically engineered P. rhodozyma would be cultivated in anappropriate medium and evaluated in its productivity of carotenoids,especially astaxanthin. A hyper producer of astaxanthin thus selectedwould be confirmed in view of the relationship between its productivityand the level of gene or protein expression which is introduced by sucha genetic engineering method.

The present invention is further illustrated with Examples describedbelow.

The following materials and methods employed in the Examples aredescribed below:

Strains

P. rhodozyma ATCC96594 (re-deposited under the accession No. ATCC 74438on Apr. 8, 1998 pursuant to the Budapest Treaty)

E. coli DH5α: F⁻, φ80d, lacZΔM15, Δ(lacZYA-argF)U169, hsd (r_(K) ⁻,m_(K) ⁺), recA1, endA1, deoR, thi-1, supE44, gyrA96, relA1 (Toyobo,Osaka, Japan)

E. coli XL1-Blue MRF′: Δ(mcrA)183, Δ(mcrCB-hsdSMR-mrr)173, endA1,supE44, thi-1, recA1, gyrA96, relA1, lac [F′ proAB, lacIqZΔM15, Tn10(tet^(r))] (Stratagene, La Jolla, USA)

E. coli SOLR: e14-(mcrA), Δ(mcrCB-hsdSMR-mrr)171, sbcC, recB, recJ,umuC::Tn5(kan^(r)), uvrC, lac, gyrA96, relA1, thi-1, endA1, ΔR, [F′proAB, lacIqZ ΔM15] Su-(nonsuppressing) (Stratagene)

E. coli TOP10: F-, mcrA, Δmrr-hsdRMS-mcrBC), φ80, ΔlacZ M15, ΔlacX74,recA1, deoR, araD139, (ara-leu)7697, galU, galK, rpsL (Str^(r)), endA1,nupG (Invitrogen, Carlsbad, USA)

Vectors

λZAPII (Stratagene)

pBluescriptII KS- (Stratagene)

pMOSBlue T-vector (Amersham, Buckinghamshire, U.K.)

pCR2.1-TOPO (Invitrogen)

Media

P. rhodozyma strain was maintained routinely in YPD medium (DIFCO,Detroit, U.S.A.).

E. coli strain was maintained in LB medium (10 g Bacto-trypton, 5 gyeast extract (DIFCO) and 5 g NaCl per liter). NZY medium (5 g NaCl, 2 gMgSO₄-7H₂O, 5 g yeast extract (DIFCO), 10 g NZ amine type A (WAKO,Osaka, Japan) per liter) is used for λ phage propagation in a soft agar(0.7% agar (WAKO)). When an agar medium was prepared, 1.5% of agar(WAKO) was supplemented.

Methods

Restriction enzymes and T4 DNA ligase were purchased from Takara Shuzo(Ohtsu, Japan).

Isolation of a chromosomal DNA from P. rhodozyma was performed by usingQIAGEN Genomic Kit (QIAGEN, Hilden, Germany) following the protocolsupplied by the manufacturer. Mini-prep of plasmid DNA from transformedE. coli was performed with the Automatic DNA isolation system (PI-50,Kurabo, Co. Ltd., Osaka, Japan). Midi-prep of plasmid DNA from an E.coli transformant was performed by using QIAGEN column (QIAGEN).Isolation of λ DNA was performed by Wizard lambda preps DNA purificationsystem (Promega, Madison, U.S.A.) following the protocol prepared by themanufacturer. A DNA fragment was isolated and purified from agarose byusing QIAquick or QIAEX II (QIAGEN). Manipulation of λ phage derivativeswas followed by the protocol prepared by the manufacturer (Stratagene).Isolation of total RNA from P. rhodozyma was performed with the phenolmethod by using Isogen (Nippon Gehe; Toyama, Japan): mRNA was purifiedfrom total RNA thus obtained by using mRNA separation kit (Clontech).cDNA was synthesized by using CapFinder cDNA construction kit(Clontech).

In vitro packaging was performed by using Gigapack III gold packagingextract (Stratagene).

The polymerase chain reaction (PCR) was performed with the thermalcycler from Perkin Elmer model 2400. Each PCR condition is described inexamples. PCR primers were purchased from a commercial supplier.Fluorescent DNA primers for DNA sequencing were purchased fromPharmacia. DNA sequencing was performed with the automated fluorescentDNA sequencer (ALFred, Pharmacia).

Competent cells of DH5α were purchased from Toyobo (Japan).

Example 1 Isolation of mRNA from P. rhodozyma and Construction of cDNALibrary

To construct cDNA library of P. rhodozyma, total RNA was isolated byphenol extraction method right after the cell disruption and the mRNAfrom P. rhodozyma ATCC96594 strain was purified by using mRNA separationkit (Clontech).

At first, Cells of ATCC96594 strain from 10 ml of two-day-culture in YPDmedium were harvested by centrifugation (1500×g for 10 min.) and washedonce with extraction buffer (10 mM Na-citrate/HCl (pH 6.2) containing0.7 M KCl). After suspending in 2.5 ml of extraction buffer, the cellswere disrupted by French press homogenizer (Ohtake Works Corp., Tokyo,Japan) at 1500 kgf/cm2 and immediately mixed with two times of volume ofisogen (Nippon gene) according to the method specified by themanufacturer. In this step, 400 μg of total RNA was recovered.

Then, this total RNA was purified by using mRNA separation kit(Clontech) according to the method specified by the manufacturer.Finally, 16 μg of mRNA from P. rhodozyma ATCC96594 strain was obtained.

To construct cDNA library, CapFinder PCR cDNA construction kit(Clontech) was used according to the method specified by themanufacturer. One μg of purified mRNA was applied for a first strandsynthesis followed by PCR amplification. After this amplification byPCR, 1 mg of cDNA pool was obtained.

Example 2 Cloning of a Partial ACC (acetyl-CoA carboxylase) Gene from P.rhodozyma

To clone a partial ACC gene from P. rhodozyma, a degenerate PCR methodwas exploited. Species and accession number to database whose sequencefor acetyl-CoA carboxylase were used for multiple alignment analysis areas follows. Arabidopsis thaliana D34630 (DDBJ) Emericella nidulansY15996 (EMBL) Gallus gallus P11029 (Swiss-Prot) Glycine max L48995(GenBank) Homo sapiens S41121 (PIR) Medicago sativa L25042 (GenBank)Ovis aries Q28559 (Swiss-Prot) Rattus norvegicus P11497 (Swiss-Prot)Saccharomyces cerevisiae Q00955 (Swiss-Prot) Schizosaccharomyces pombeP78820 (Swiss-Prot) Ustilago maydis S49991 (PIR)

Two mixed primers whose nucleotide sequences were designed andsynthesized based on the common sequence of known acetyl-CoA carboxylasegenes from other species: acc9 (sense primer) (SEQ ID NO:4) and acc13(antisense primer) (SEQ ID NO:5) (in the sequences “n” means nucleotidesa, c, g or t, “h” means nucleotides a, c or t, “m” means nucleotides aor c, “k” means nucleotides g or t, and “y” means nucleotides c or t).After the PCR reaction of 25 cycles of 95° C. for 30 seconds, 45° C. for30 seconds and 72° C. for 15 seconds by using ExTaq (Takara Shuzo) as aDNA polymerase and cDNA pool obtained in Example 1 as a template,reaction mixture was applied to agarose gel electrophoresis. One PCRband that had a desired length (0.8 kb) was recovered from the agarosegel and purified by QIAquick (QIAGEN) according to the method by themanufacturer and then ligated to pMOSBlue-T-vector (Amersham). Aftertransformation of competent E. coli DH5α, 6 white colonies were selectedand plasmids were isolated with Automatic DNA isolation system. As aresult of sequencing, it was found that 3 clones had a sequence whosededuced amino acid sequence was similar to known acetyl-CoA carboxylasegenes. These isolated cDNA clones were designated as pACC1014 and usedfor further screening study.

Example 3 Isolation of Genomic DNA from P. rhodozyma

To isolate a genomic DNA from P. rhodozyma, QIAGEN genomic kit was usedaccording to the method specified by the manufacturer.

At first, cells of P. rhodoyma ATCC96594 strain from 100 ml of overnightculture in YPD medium were harvested by centrifigation (1500×g for 10min.) and washed once with TE buffer (10 mM Tris/HCl (pH 8.0) containing1 mM EDTA). After suspending in 8 ml of Y1 buffer of the QIAGEN genomickit, lyticase (SIGMA, St. Louis, U.S.A.) was added at the concentrationof 2 mg/ml to disrupt cells by enzymatic degradation and the reactionmixture was incubated for 90 min at 30° C. and then proceeded to thenext extraction step.

Finally, 20 μg of genomic DNA was obtained.

Example 4 Southern Blot Hybridization by Using pACC1014 as a Probe

Southern blot hybridization was performed to clone a genomic fragmentwhich contains ACC gene from P. rhodozyma. Two μg of genomic DNA wasdigested by EcoRI and subjected to agarose gel electrophoresis followedby acidic and alkaline treatment. The denatured DNA was transferred tonylon membrane (Hybond N+, Amersham) by using transblot (Joto Rika,Tokyo, Japan) for an hour. The DNA which was transferred to nylonmembrane was fixed by a heat treatment (80° C., 90 min). A probe wasprepared by labeling a template DNA (EcoRI and SalI-digested pACC1014)with DIG multipriming method (Boehringer Mannheim). Hybridization wasperformed with the method specified by the manufacturer. As a result, ahybridized band was visualized in the range from 2.0 to 2.3 kilobases(kb).

Example 5 Cloning of a Genomic Fragment Containing the ACC Gene

4 μg of the genomic DNA were digested by EcoRI and subjected to agarosegel electrophoresis. Then, DNAs with a length within the range from 1.5to 2.7 kb was recovered by QIAEX II gel extraction kit (QIAGEN)according to the method specified by the manufacturer. The purified DNAwas ligated to 0.5 μg of EcoRI-digested and CIAP (calf intestinealkaline phosphatase)-treated λZAP II (Stratagene) at 16° C. overnight,and packaged by Gigapack III gold packaging extract (Stratagene). Thepackaged extract was infected to E. coli MRF′ strain and over-laid withNZY medium poured onto LB agar medium. About 5000 plaques were screenedby using EcoRI and SalI-digested pACC1014 as a probe. Five plaques werehybridized to the labeled probe.

The in vivo excision protocol was applied to these λZAP II derivativescontaining putative ACC gene from P. rhodozyma by following theinstruction manual (Stratagene) to clone the insert fragment into E.coli cloning vector, pBluescript SK. Each clone recovered from fivepositive plaques was subjected for sequencing analysis and it was foundthat the three of them had the identical sequence to the insert fragmentof pACC1014. One of the clone was named as pACC1224 and used for furtherstudy. As a result of whole sequencing of the entire region of insertfragment in pACC1224, it was suggested that this clone contained neitherits 5′- nor 3′-end of the ACC gene.

Example 6 Cloning of the Flanking Region of the Insert Fragment inpACC1224 from the Genome of P. rhodozyma by Genome Walking Method

Two PCR primers were synthesized based on the internal sequence ofpACC1224 and used for the genome walking method: acc17 (SEQ ID NO:6) andacc18 (SEQ ID NO:7). The protocol of the instruction manual providedfrom the supplier (Clontech) was followed for the genome walking method.In the PCR reaction using acc17 primer, a 2.8 kb PCR band emerged fromthe genomic StuI library. In the case of acc18 primer, a 2.2 kb PCR bandwas produced in the genomic PvuII library. These PCR bands were clonedinto pCR2.1-TOPO (Invitrogen) and it was revealed that 2.8 kb PCR bandcontained a 5′ fragment of ACC gene and 2.2 kb PCR band contained 3′fragment of ACC gene, respectively. The clones containing 2.8 kb and 2.2kb PCR fragment were named as pACCStu107 and pACCPvd107, respectivelyand used for further study.

Example 7 Southern Blot Hybridization by Using pACCStu107 and pACCPvd107as Probes

Southern blot hybridization was performed to clone a genomic fragmentwhich covered the ACC gene from P. rhodozyma. 2 μg of genomic DNA wasdigested by EcoRI and subjected to agarose gel electrophoresis followedby acidic and alkaline treatment. The denatured DNA was transferred tonylon membrane (Hybond N+, Amersham) by using transblot (Joto Rika,Tokyo, Japan) for an hour. The DNA which was transferred to nylonmembrane was fixed by a heat treatment (80° C., 90 min). A probe wasprepared by labeling a template DNA (EcoRI-digested pACCStu107 andpACCPvd107) with the DIG multi-priming method (Boehringer Mannheim).Hybridization was performed with the method specified by themanufacturer. As a result, several hybridized bands whose size was closeto 2.0 kb, 0.9 kb and 0.6 kb were visualized when the insert fragment inpACCStu107 was used as a probe. In the case that the insert fragment inpACCPvd107 was used as a probe, a hybridized band was visualized in therange from 6.0 kb to 6.5 kb.

Example 8 Cloning of the Genomic Clone Covering the ACC Gene

In a similar manner to Example 5, the genomic fragment containing theinsert fragment in pACCStu107 and pACCPvd107 was cloned by plaquehybridization. 4 μg of the genomic DNA was digested by EcoRI andsubjected to agarose gel electrophoresis. Then, DNAs with a lengthwithin the following range were recovered by QIAEX II gel extraction kit(QIAGEN) according to the method specified by the manufacturer: (1) from2.7 to 5.0 kb; (2) from 1.4 to 2.7 kb; and (3) from 0.5 to 1.4 kb.

Each purified DNA was ligated to 0.5 μg of EcoRI-digested and CIAP (calfintestine alkaline phosphatase)-treated λZAP II (Stratagene) at 16° C.overnight, and packaged by Gigapack III gold packaging extract(Stratagene). The packaged extract was infected to E. coli MRF′ strainand over-laid with NZY medium poured onto LB agar medium. About 5000plaques were screened by using EcoRI-digested pACCStu107 and pACCPvd107as probes.

The following candidates were isolated after plaque hybridization study.

1) 3 plaques from the 2.7 to 6.0 kb library by using the insert ofpACCPvd107 as a probe.

2) 3 plaques from the 1.4 to 2.7 kb library by using the insert ofpACCStu107 as a probe.

3) 21 plaques from the 0.5 to 1.4 kb library by using the insert ofpACCStu107 as a probe.

The in vivo excision protocol was applied to these λZAP II derivativescontaining putative ACC gene from P. rhodozyma by following theinstruction manual (Stratagene) to clone the insert fragment into E.coli cloning vector, pBluescript SK. Each clone recovered from thepositive plaques was subjected for sequencing analysis. At least eachclone had the putative ACC gene from BLAST X analysis(http://www.blast.genome.ad.jp/). The following clones were selected andused for further analysis:

pACC119-18 having a 6 kb insert and covering the 3′ end of the ACC gene;

pACC119-17-0.6 having a 0.6 kb insert flanking the 5′ end of thepACC1224 insert fragment;

pACC119-17-2 having a 2 kb insert flanking the 5′ end of thepACC119-17-0.6 insert fragment; and

pACC127-17-0.9 having a 0.9 kb insert flanking the 5′ end of thepACC119-17-2 insert fragment.

As a result of whole sequencing of the entire region of insert fragmentin pACC119-18, pACC119-17-0.6, pACC119-17-2 and pACC127-17-0.9, it wassuggested that these clones did not cover the 5′ end of the ACC gene.

Example 9 Cloning of the Franking Region of the Insert Fragment inpACC127-17-0.9 from the Genome of P. rhodozyma by Genome Walking Method

PCR primer acc26 (SEQ ID NO:8) was synthesized based on the internalsequence of pACC127-17-0.9 and used for genome walking method.

In the PCR reaction using acc26 primer, a 2.6 kb PCR band emerged fromthe genomic PvuII library. This PCR band was cloned into pCR2.1-TOPO(Invitrogen) and it was revealed that this clone contained 5′ fragmentof ACC gene as a result of BLAST X analysis. This clone was named aspACCPvu126 and used for further study.

Example 10 Southern Blot Hybridization by using pACCPvu126 as a Probe

Southern blot hybridization was performed to clone a genomic fragmentwhich covered 5′ end of ACC gene from P. rhodozyma. In a similar manneras Example 7, Southern blot hybridization was performed. A probe wasprepared by labeling a template DNA (EcoRI-digested pACCPvu116) with DIGmultipriming method (Boehringer Mannheim). Hybridization was performedwith the method specified by the manufacturer. As a result, a hybridizedband whose size was close to 5.0 kb was visualized.

Example 11 Cloning of the Genomic Clone Covering 5′ End of ACC Gene

In a similar manner to Example 8, the genomic fragment containing theinsert fragment in pACCPvu126 was cloned by plaque hybridization. Thegenomic library covering 2.7 to 6.0 kb in length prepared in Example 8was also used. Twelve positive plaques which hybridized to the insertfragment of pACCPvu126 labeled with DIG were isolated and subjected toin vivo excision to obtain plasmid DNA. As a result of sequencing forthus isolated plasmids, most of the plasmids had the identical sequenceto the insert fragment of pACCPvu126. One of the clones was named aspACC204 and used for further study.

Example 12 Cloning of the Gapped Region Between pACC204 andpACC127-17-0.9

As a result of BLAST X analysis against known acetyl-CoA carboxylasegenes succeeding to the sequencing study of 3′ end of the insertfragment in pACC204 and 5′ end of the insert fragment in pACC127-17-0.9,it was suggested that an approximately 0.3 kb fragment could be stillmissing for a coverage of the entire ACC gene. The following PCR primerswere synthesized based on the internal sequence of pACC204 andpACC127-17-0.9: acc43 (sense primer) (SEQ ID NO:9) and acc44 (antisenseprimer) (SEQ ID NO:10). After the PCR reaction of 25 cycles of 94° C.for 15 seconds, 55° C. for 30 seconds and 72° C. for 15 seconds by usingHF polymerase (Clontech) as a DNA polymerase and a genomic DNA obtainedin Example 3 as a template, the reaction mixture was applied to agarosegel electrophoresis. One PCR band that had a desired length (0.3 kb) wasrecovered from the agarose gel and purified by QIAquick (QIAGEN)according to the method by the manufacturer and then cloned intopCR2.1-TOPO (Invitrogen). After transformation of competent E. coli TOP10, 6 white colonies were selected and plasmids were isolated withAutomatic DNA isolation system. As a result of sequencing, it was foundthat 5 clones had an identical sequence from each other. One of theisolated clones was designated as pACC210.

Example 13 Sequencing of a Complete Genomic Fragment Containing ACC Gene

pACC204, pACC210, pACC127-17-0.9, pACC119-17-2, pACC119-17-0.6, pACC1224and pACC119-18 were sequenced with primer walking procedure by usingAutoRead sequencing kit (Pharmacia).

As a result of sequencing, the nucleotide sequence comprising 10561 basepairs of the genomic fragment containing the ACC gene from P. rhodozymacontaining its promoter (1445 base pairs) and terminator (1030 basepairs) was determined (SEQ ID NO:1).

The coding region was 8086 base pairs long and consisted of 19 exons and18 introns. Introns were dispersed all through the coding region without5′ or 3′ bias. It was found that an open reading frame (SEQ ID NO:2)consists of 2187 amino acids (SEQ ID NO:3) whose sequence is strikinglysimilar to the known amino acid sequence of acetyl-CoA carboxylase fromother species (56.28% identity to acetyl-CoA carboxylase from Emericellanidulans) as a result of homology search by GENETYX-SV/RC software(Software Development Co., Ltd., Tokyo, Japan).

FIG. 1 depicts a cloned DNA fragment covering ACC gene region on thechromosome of P. rhodozyma.

Example 14 Construction of Antisense Plasmid for ACC Gene

An antisense gene fragment which covers the entire structure gene forACC gene is amplified by PCR and then cloned into an integration vectorin which the antisense ACC gene is transcribed by its own ACC promoterin P. rhodozyma.

The primers include an asymmetrical recognition sequence for therestriction enzyme, SfiI (GGCCNNNNNGGCC) but their asymmetricalhang-over sequence is designed to be different. This enables adirectional cloning into expression vector which has the sameasymmetrical sequence at their ligation sequence. The use of such aconstruction is disclosed in EP 1,158,051.

For the promoter and terminator fragment which can drive thetranscription of the antisense ACC gene, the ACC promoter and terminatoris cloned from the chromosome by using the sequence information listedin SEQ ID NO:1. The ACC terminator fragment is fused to a G418 resistantcassette by ligating the DNA fragment containing the ACC terminator to aG418 resistant cassette of pG418Sa330 (EP 1,035,206) to an appropriatevector such as pBluescriptII KS-(Stratagene).

Then, 3.1 kb of the SacI fragment containing ribosomal DNA (rDNA) locus(Wery et al., Gene, 184, 89-97, 1997) is inserted downstream of the G418cassette on thus prepared plasmid. The rDNA fragment exists inmulticopies on the chromosome of eukaryote. The integration event viathe rDNA fragment would result in multicopied integration onto thechromosome of the host used and this enables the overexpression offoreign genes which are harbored in expression vector.

Subsequently, ACC promoter is inserted in the upstream of ACC terminatorto construct of expression vector which functions in P. rhodozyma.

Finally, the antisense ACC construct is completed by inserting the 1.5kb of SfiI fragment containing antisense ACC into thus preparedexpression vector functioning in P. rhodozyma. A similar plasmidconstruction is disclosed in EP 1,158,051.

Example 15 Transformation of P. rhodozyma with an ACC-Antisense Vector

The ACC-antisense vector thus prepared is transformed into P. rhodozymawild type strain, ATCC96594. The protocol for the biolistictransformation is disclosed in EP 1,158,051.

Example 16 Characterization of Antisense ACC Recombinant of P. rhodozyma

Antisense ACC recombinant of P. rhodozyma, ATCC96594 is cultured in 50ml of YPD medium in 500 ml Erlenmeyer flask at 20° C. for 3 days byusing their seed culture which grows in 10 ml of YPD medium in testtubes (21 mm in diameter) at 20° C. for 3 days. For analysis ofcarotenoid produced appropriate volume of culture broth is withdrawn andused for analysis of their growth, productivity of carotenoids,especially astaxanthin. For analysis of growth, optical density at 660nm is measured by using a UV-1200 photometer (Shimadzu Corp., Kyoto,Japan) in addition to the determination of their dried cell mass bydrying up the cells derived from 1 ml of broth after microcentrifugationat 100° C. for one day. For the analysis of the content of astaxanthinand total carotenoids, cells are harvested from 1.0 ml of broth aftermicrocentrifugation and used for the extraction of the carotenoids fromcells of P. rhodozyma by disruption with glass beads. After extraction,disrupted cells are removed by centrifugation and the resultant isanalyzed for carotenoid content with HPLC. The HPLC condition used is asfollows: HPLC column: Chrompack Lichrosorb si-60 (4.6 mm, 250 mm),Temperature: room temperature, Eluent: acetone/hexane (18/82) add 1 ml/Lof water to eluent, Injection volume: 10 μl, Flow rate: 2.0 ml/min,Detection: UV at 450 nm. A reference sample of astaxanthin can beobtained from Hoffmann La-Roche (Basel, Switzerland).

FIG. 1 depicts a deducted biosynthetic pathway from acetyl-CoA toastaxanthin in P. rhodozyma.

FIG. 2 depicts a cloned DNA fragment covering ACC gene region on thechromosome of P. rhodozyma.

1. An isolated polynucleotide comprising a nucleic acid molecule one ormore selected from the group consisting of: (a) nucleic acid moleculesencoding at least the mature form of the polypeptide depicted in SEQ IDNO:3; (b) nucleic acid molecules comprising the coding sequence asdepicted in SEQ ID NO:2; (c) nucleic acid molecules whose nucleotidesequence is degenerate as a result of the genetic code to a nucleotidesequence of (a) or (b); (d) nucleic acid molecules encoding apolypeptide derived from the polypeptide encoded by a polynucleotide of(a) to (c) by way of substitution, deletion and/or addition of one orseveral amino acids of the amino acid sequence of the polypeptideencoded by a nucleotide of (a) to (c); (e) nucleic acid moleculesencoding a polypeptide derived from the polypeptide whose sequence hasan identity of 56.3% or more to the amino acid sequence of thepolypeptide encoded by a nucleic acid molecule of (a) or (b); (f)nucleic acid molecules comprising a fragment encoded by a nucleic acidmolecule of any one of (a) to (e) and having acetyl-CoA carboxylaseactivity; (g) nucleic acid molecules comprising a polynucleotide havinga sequence of a nucleic acid molecule amplified from a Phaffia nucleicacid library using the primers depicted in SEQ ID NO:4, 5, and 6; (h)nucleic acid molecules encoding a polypeptide having acetyl-CoAcarboxylase activity, wherein said polypeptide is a fragment of apolypeptide encoded by any one of (a) to (g); (i) nucleic acid moleculescomprising at least 15 nucleotides of a polynucleotide of any one of (a)to (d); (j) nucleic acid molecules encoding a polypeptide havingacetyl-CoA carboxylase activity, wherein said polypeptide is recognizedby antibodies that have been raised against a polypeptide encoded by anucleic acid molecule of any one of (a) to (h); (k) nucleic acidmolecules obtainable by screening an appropriate library under stringentconditions with a probe having the sequence of the nucleic acid moleculeof any one of (a) to (j), and encoding a polypeptide having acetyl-CoAcarboxylase activity; (l) nucleic acid molecules whose complementarystrand hybridizes under stringent conditions with a nucleic acidmolecule of any one of (a) to (k), and encoding a polypeptide havingacetyl-CoA carboxylase activity.
 2. An isolated polynucleotidecomprising a nucleic acid molecule one or more selected from the groupconsisting of: (m) nucleic acid molecules comprising the nucleotidesequence as depicted in SEQ ID NO:1; (n) nucleic acid molecules whosenucleotide sequence is degenerate as a result of the genetic code to anucleotide sequence of (m); (o) nucleic acid molecules encoding apolypeptide derived from the polypeptide encoded by a polynucleotide of(m) or (n) by way of substitution, deletion and/or addition of one orseveral amino acids of the amino acid sequence of the polypeptideencoded by a nucleotide of (m) or (n); (p) nucleic acid moleculesencoding a polypeptide derived from the polypeptide whose sequence hasan identity of 56.3% or more to the amino acid sequence of thepolypeptide encoded by a nucleic acid molecule of (m); (q) nucleic acidmolecules comprising a fragment encoded by a nucleic acid molecule ofany one of (m) to (p) and having acetyl-CoA carboxylase activity; (r)nucleic acid molecules comprising a polynucleotide having a sequence ofa nucleic acid molecule amplified from a Phaffia nucleic acid libraryusing the primers depicted in SEQ ID NO: 4, 5, and 6; (s) nucleic acidmolecules encoding a polypeptide having acetyl-CoA carboxylase activity,wherein said polypeptide is a fragment of a polypeptide encoded by anyone of (m) to (r); (t) nucleic acid molecules comprising at least 15nucleotides of a polynucleotide of any one of (m) to (o); (u) nucleicacid molecules encoding a polypeptide having acetyl-CoA carboxylaseactivity, wherein said polypeptide is recognized by antibodies that havebeen raised against a polypeptide encoded by a nucleic acid molecule ofany one of (m) to (s); (v) nucleic acid molecules obtainable byscreening an appropriate library under stringent conditions with a probehaving the sequence of the nucleic acid molecule of any one of (m) to(u), and encoding a polypeptide having acetyl-CoA carboxylase activity;(w) nucleic acid molecules whose complementary strand hybridizes understringent conditions with a nucleic acid molecule of any one of (m) to(v), and encoding a polypeptide having acetyl-CoA carboxylase activity.3. The isolated polynucleotide of claim 1, wherein said polynucleotideencodes amino acid sequence which is identified by SEQ ID NO:3 or hasidentity of 56.3% or more with SEQ ID NO:
 3. 4. The isolatedpolynucleotide of claim 1, wherein said polynucleotide is derived from astrain of P. rhodozyma or Xanthophylomyces dendrorhous.
 5. A method formaking a recombinant vector comprising inserting the polynucleotide ofclaim 1 into a vector.
 6. A recombinant vector containing thepolynucleotide of claim
 1. 7. The vector of claim 6 in which thepolynucleotide of claim 1 is operatively linked to expression controlsequences allowing expression in prokaryotic or eukaryotic cells.
 8. Amethod of making a recombinant organism comprising introducing thevector of claim 6 into a host organism.
 9. The method of claim 8,wherein said host organism is selected from E. coli, baculovirus, or S.cerevisiae.
 10. The recombinant organism containing the vector of claim6.
 11. A process for producing a polypeptide having acetyl-CoAcarboxylase activity comprising culturing the recombinant organism ofclaim 10 and recovering the polypeptide from the culture of saidrecombinant organism.
 12. A polypeptide obtainable by the process ofclaim
 11. 13. An antibody that binds specifically to the polypeptide ofclaim
 12. 14. An antisense polynucleotide against the polynucleotide ofclaim
 1. 15. A method for making a recombinant vector comprisinginserting the polynucleotide of claim 14 into a vector.
 16. Arecombinant vector containing the polynucleotide of claim
 14. 17. Thevector of claim 16 in which the polynucleotide of claim 14 isoperatively linked to expression control sequences allowing expressionin prokaryotic or eukaryotic cells.
 18. A method of making a recombinantorganism comprising introducing the vector of claim 16 into a hostorganism.
 19. The method of claim 18, wherein said host organism belongsto a strain of Phaffia rhodozyma or Xanthophylomyces dendrorhous. 20.The recombinant organism containing the vector of claim
 16. 21. Therecombinant organism of claim 20, wherein said organism is characterizedin that whose gene expression of acetyl-CoA carboxylase is reducedcompared to the host organism, thereby is capable of producingcarotenoids in an enhanced level relative to a host organism.
 22. Therecombinant organism according to claim 21, wherein the gene expressionof acetyl-CoA carboxylase is reduced by means of the technique selectedfrom antisense technology, site-directed mutagenesis, error prone PCR,or chemical mutagenesis.
 23. A process for producing carotenoids, whichcomprises cultivating the recombinant organism of claim
 21. 24. Theprocess of claim 23, wherein said carotenoids are selected one or morefrom astaxanthin, β-carotene, lycopene, zeaxanthin, canthaxanthin. 25.The process according to claim 23, wherein the gene expression ofacetyl-CoA carboxylase is reduced in the recombinant organism of claim21 by means of the technique selected from antisense technology,site-directed mutagenesis, error prone PCR, or chemical mutagenesis. 26.A process for the production of a carotenoid by culturing amicroorganism under suitable conditions and, optionally, recovering theresulting carotenoid, wherein the microorganism is characterized in thatits gene expression of acetyl-CoA carboxylase is reduced, e.g. by meansof the technique selected from antisense technology, site-directedmutagenesis, error prone PCR, or chemical mutagenesis.
 27. The isolatedpolynucleotide of claim 2, wherein said polynucleotide encodes aminoacid sequence which is identified by SEQ ID NO: 3 or has identity of56.3% or more with SEQ ID NO:
 3. 28. The isolated polynucleotide ofclaim 2, wherein said polynucleotide is derived from a strain of P.rhodozyma or Xanthophylomyces dendrorhous.
 29. A method for making arecombinant vector comprising inserting the polynucleotide of claim 2into a vector.
 30. A recombinant vector containing the polynucleotide ofclaim
 2. 31. The vector of claim 30 in which the polynucleotide of claim2 is operatively linked to expression control sequences allowingexpression in prokaryotic or eukaryotic cells.
 32. A method of making arecombinant organism comprising introducing the vector of claim 30 intoa host organism.
 33. The method of claim 32, wherein said host organismis selected from E. coli baculovirus, or S. cerevisiae.
 34. Therecombinant organism containing the vector of claim
 30. 35. A processfor producing a polypeptide having acetyl-CoA carboxylase activitycomprising culturing the recombinant organism of claim 34 and recoveringthe polypeptide from the culture of said recombinant organism.
 36. Apolypeptide obtainable by the process of claim
 35. 37. An antibody thatbinds specifically to the polypeptide of claim
 36. 38. An antisensepolynucleotide against the polynucleotide of claim
 2. 39. A method formaking a recombinant vector comprising inserting the polynucleotide ofclaim 38 into a vector.
 40. A recombinant vector containing thepolynucleotide of claim
 38. 41. The vector of claim 40 in which thepolynucleotide of claim 38 is operatively linked to expression controlsequences allowing expression in prokaryotic or eukaryotic cells.
 42. Amethod of making a recombinant organism comprising introducing thevector of claim 40 into a host organism.
 43. The method of claim 42,wherein said host organism belongs to a strain of Phaffia rhodozyma orXanthophylomyces dendrorhous.
 44. The recombinant organism containingthe vector of claim
 40. 45. The recombinant organism of claim 44,wherein said organism is characterized in that whose gene expression ofacetyl-CoA carboxylase is reduced compared to the host organism, therebyis capable of producing carotenoids in an enhanced level relative to ahost organism.
 46. The recombinant organism according to claim 45,wherein the gene expression of acetyl-CoA carboxylase is reduced bymeans of the technique selected from antisense technology, site-directedmutagenesis, error prone PCR, or chemical mutagenesis.
 47. A process forproducing carotenoids, which comprises cultivating the recombinantorganism of claim
 45. 48. The process of claim 47, wherein saidcarotenoids are selected one or more from astaxanthin, β-carotene,lycopene, zeaxanthin, canthaxanthin.
 49. The process according to claim47, wherein the gene expression of acetyl-CoA carboxylase is reduced inthe recombinant organism of claim 45 by means of the technique selectedfrom antisense technology, site-directed mutagenesis, error prone PCR,or chemical mutagenesis.